Organic phosphate mineralization by Bacillus sphaericus and Pseudomonas cepacia

Journal of Agricultural Technology 2014 Vol. 10(4): 913-922 Available online http://www.ijat-aatsea.com Journal of Agricultural Technology 2014, Vol. ISSN 10(4): 1686-9141 915-922 Organic phosphate mineralization by Bacillus sphaericus and Pseudomonas cepacia Vimal Ramani * Dairy Science College, Kamdhenu University, Amreli -365601. Gujarat. India Vimal Ramani (2014) Organic phosphate mineralization by Bacillus sphaericus and Pseudomonas cepacia. Journal of Agricultural Technology 10(4):915-922. Phosphorus (P) content of average soils is about 0.05% (w/w) but only about 0.1% of this P is available to plants. On the other hand an average utilization of added fertilizer P by the plants ranges from 15 to 25 % only, due to its fixation in soil. In order to become available to plants, organic P compounds must first be hydrolyzed by phosphatases. In soil, this process is predominantly mediated by the activity of soil microorganisms, and plant roots. In view of its wide spread deficiency in the soil, its low availability, high fixing tendency and high cost of industrial phosphate fertilizers the seed/soil inoculation with phosphate solubilizing microorganisms (PSMs) has a profound significance in the modern day of agriculture. Intensive screening of phosphate solubilizing bacteria with genetic potential for increased tolerance to high salt, high ph and high temperature could enhance production of food and forage in semi-arid regions. Emphasizing particularly on this hypothesis 165 phosphate solubilizing bacteria were isolated. Among these, two cultures Bacillus sphaericus and Pseudomonas cepacia were selected on the basis of salt tolerance property and PS activity with different forms of phosphates. In the present study both the bacterial culture were explored for their organic phosphate mineralization ability. Both the isolates were showed variable capability to mineralize various forms of organic phosphate. This organic P mineralizing ability can be profited to the village farmers by its successful use along with green manures, crop residues and recyclable waste. Keywords: Phosphate mineralization, Microbial inoculants, Phosphate solubilizing bacteria, Bacillus sphaericus, Pseudomonas cepacia. Introduction Because of its complex nature, the organic fraction of soil P has received relatively little attention. This fraction may accounts 50 to 80% of the soil-p, depending upon the nature of soil and its composition (Dalal, 1978; McLoughlin et al., 1990). Vegetation and decaying plant residues entering the soil are the main sources of organic phosphorus compounds. The soil organic P fraction is composed chiefly of phytin, phospholipids, nucleic acids and their derivatives. Phytin and its derivatives are probably the main organic P component (Pederson, 1953). The nucleic acid comprises not more than 5% of the organic P (Adams et al., 1954). The * Corresponding author: Vimal M. Ramani; e-mail: vimalmramani@gmail.com 915

amount of lecithin in the soil is small, possibly about 1% of the total soil P. The remaining of the soil organic P occurs in unknown forms. Phytic acid, an organic phosphate form is of special interest to plants, because it is the principal storage form of phosphorus in mature seeds of both monocoat and dicoat plants. Phytin is a calcium magnesium salt of phytic acid (inositol phosphate). Inositol phosphate may have one to six phosphorus atoms per inositol unit. On germination, phosphate from phytin becomes available for the phopsphorylation reaction of the seedling. Phospholipids include lecithin and cephalin in which phosphate is esterified with a nitrogenous base. Lecithin is made up of glycerol, fatty acids, phosphate and choline. In spite of relative abundance of P in nature, phosphate is sometimes a growth limiting factor for soil microorganisms also, because most of its natural supply occurs as insoluble salts. To cope up with this P limitations, most bacteria (Au et al., 1991), yeast and fungi (Furukawa et al., 1987; Goldman et al., 1987) produces extracellular phosphatases. Phosphatases are group of enzymes that catalyze the hydrolysis of both esters and anhydrates of H 3 PO 4. These enzymes are classified as acid and alkaline phosphatases because their maximum activities occur at low (ph 6.5) and high (ph 11) ph ranges. Acid phosphatases are produced by both, microorganisms and plant, but alkaline phosphatases are produced mainly by microorganisms. Mineralization of organic compounds like nucleic acids and lecithin was carried out by nuclease and lecithinase respectively under favorable conditions. Phytase, breaks the phytic acid to myoinositol and phosphate via intermediate inositol polyphosphate. (Abul et al., 1990) Among microorganisms the phytase enzyme has been studied most extensively in Aspergilli species. Phytase activity is also known to be widespread in Penicillium, Cunninghamella, Arthrobacter, Streptomyces. Proteus, Serratia, Pseudomonas, and Bacillus are known to have ability. The ribonucleases and deoxyribonucleases are synthesized by certain species of Aspergillus, Penicillium, Strepromyces, Achromobacter and Clsotridium. Lecithinase produced by bacteria including actinomycetes and fungi. Somani and Saxena (1971, 1975, 1976) also reported the reduction in the content of organic phosphorus in soil was associated with simultaneous increase in the numbers of microorganisms capable of dephosphorylating these substances, which show their involvement in the mineralization process. In the present investigation Bacillus sphaericus and Burkholderia cepacia were evaluated for their organic phosphate mineralizing ability. Materials and methods Organisms: B. sphaericus and B. cepacia maintained on Pikovskaya s agar slants were used. 916

Journal of Agricultural Technology 2014, Vol. 10(4): 915-922 Culture Media: Modified Pikovskaya s broth: TCP in Pikovskaya s broth was replaced with 0.5% each of deoxyribonucleic acid(), ribonucleic acid(), sodium-β-glycerophosphate (SGP), calcium phytate(cp), sodium phytate(sp) and adenosine triphosphate (). Nutrient broth: The medium was used to prepare bacterial inoculum. The compositions of Pikovskaya s broth and N. broth are mentioned in chapter 2. All media were sterilized by autoclaving for 15 minutes at 15 lb p.s.i (121 C). Preparation of an inoculum: An inoculum of each culture was prepared as described in chapter 2. Inoculation and growth condition Two sets, each of six flasks containing 100ml modified Pikovskaya s broth, were inoculated aseptically with 1ml inoculum in each flask. The inoculated flask were incubated at 28 ± 0.2 C for 21 d for phosphate mineralization under static condition and shaken at 12 h intervals. Respective uninoculated flasks were incubated as control. The experiment was carried out in triplicate. Phosphorus estimation and ph measurement At periodic intervals, 10 ml medium was withdrawn from each flask aseptically and centrifuged at 10,000 r.p.m. for 20 minutes. Supernatant was analysed for its water soluble -P content by chlorostannous reduced molybdophosphoric acid blue method. (Jackson, 1973) The ph of the medium was determined by Elico ph meter. Results and discussions Table 1 and 2 show soluble P released from the organic phosphorus compound and drift in the ph of the medium. B. sphaericus and B. cepacia were found to mineralize different organic phospatic compounds in variable amounts. The breakdown of organophosphates indicates the presence of phosphatases such as nuclease or phytase. Maximum P released in presence of calcium phytate by both the organisms. The hydrolysis of different organic phosphates with subsequent release of soluble P by B. sphaericus and B. cepacia can be arranged in decreasing order as follows; For B. sphaericus; CP > SGP > > > > SP (118.96) (110.38) (101.59) (74.53) (35.90) (26.70) 917

For B. cepacia; CP > SGP > > > SP > (111.11) (106.98) (85.81) (50.81) (23.01) (2) Above results clearly indicate that calcium phytate stand distinctly above among all organophosphates as the best substrate for minerlization. Dave (1999) also reported calcium phytate as the most preferred compound among different organic phosphates tested, by Psudomonas fluorescens. This observation is in affirmation with our results. However, Samaha (1998) found that SGP was mineralzed maximally by Aspergillus niger among different organic phosphates tested. B. sphaericus solubilized SGP,, and SP maximally on 21 st d and CP and on 15 th d and 9 th, respectively. In case of B. cepacia, SGP,, and SP hydrolyzed maximally on 18 th d while CP and on 9 th d and 21 st d, respectively. Table 3 shows acid phosphatases production by B. sphaericus which is distinctly higher in and containing medium in initial phase and later phase of the experiment, respectively. Among the two phytates, the enzyme production was more in calcium salt than in sodium salt of the phytic acid and among nucleic acids, double in than in. Among the six substrates, supported acid phosphatase production least. Table 4 shows alkaline phosphatase production by B. sphaericus using different organic phosphorus compounds. The order of the compounds on the basis of phophatase production was as follows; >SGP>CP>SP > = On comparing two enzymes, the organism produced more alkaline phosphatase with and SGP and more acid phosphatase with, CP and while, production of both enzymes was equal with SP. Table 5 reveals acid phosphatase production with different substrates by B. cepacia. The enzyme activities was doubled with than with. CP showed more phosphatase production than SP. Acid phosphatase production was highest in case of followed by and CP. Table 6 shows alkaline phosphatase production by B. cepacia in presence of different organic phosphorus compounds. A phosphatase activity was maximum on 21 st d with, SGP and CP and on 18 th and 15 th d with SP, and, respectively. Maximum alkaline phosphatase activity was noted with SGP followed by SP,, CP, and. Phosphatase activity was nearly equal with and. The organism showed more alkaline phosphatase activity with, SGP, CP, SP and and more acid phosphatase activities with and. On comparing two enzyme activity, maximum acid phosphatase activities was 918

Journal of Agricultural Technology 2014, Vol. 10(4): 915-922 more (with ) than that of maximum alkaline phosphatase activity (with SGP). The reduction in enzyme activity with some organic compounds tested once attaining its maxima may be due to the increase in soluble phosphorus concentration in the culture medium which repress the native phosphatase activity. Utilization of phytate by both the organisms suggests the presence of phosphatases and its beneficial roll in phosphate uptake by plants, as phytate is present in large quantity in the plants. The ph drift was observed with different organic phosphates mineralized by B. sphaericus and B. cepacia. The ph drift was more towards acidic side, ranged between ph 3.8 to 2.9 and 3.9 to 3.2 with and, respectively, compared to rest of the compounds. While, with all other organic phosphorus sources the ph drifted towards neutral to alkaline side. This is may be due to the presence of a very sensitive control mechanism for regulation of gene involved in phosphate utilization by microorganisms, which not allowing organic acid production but permit the release of extracellular acid or alkaline phosphatases in presence of different organophosphate compounds. Taking together, all above results suggest that both the isolates are also able to scavenge phosphate from organic P sources. This organic P mineralizing ability can be profited to the village farmers by its successful use along with green manures, crop residues and recyclable waste. Table 3. Acid phosphatase activity of B. sphaericus with different organic P sources Organic P sources Sodium β glycerophosphate Acid Phosphatase (EU) Days of incubation 0.45 0.33 0.70 0.50 0.29 0.69 0.50 0.24 0.25 0.65 0.52 0.29 0.42 0.80 0.38 0.61 0.82 0.26 0.58 919

Table 4. Alkaline phosphatase activity of B. sphaericus with different organic P sources Organic P sources Sodium β glycerophosphate Alkaline Phosphatase (EU) Days of incubation 0.80 1.10 1.09 1.12 0.36 1.20 0.42 0.38 1.22 0.16 1.30 0.80 0.21 Table 5. Acid phosphatase activity of B. cepacia with different organic P sources Organic P sources Sodium β glycerophosphate Acid Phosphatase (EU) Days of incubation 0.39 0.48 0.80 0.50 0.21 0.35 0.23 0.82 0.81 0.68 0.29 0.26 0.79 0.16 1.20 0.65 0.29 0.70 Table 6. Alkaline phosphatase activity of B. cepacia. with different organic P sources Organic P sources Sodium glycerophosphate β Alkaline Phosphatase (EU) Days of incubation 0.35 0.26 0.42 0.36 0.37 0.48 0.35 0.39 0.38 0.59 0.16 0.46 0.65 0.68 0.42 0.78 0.16 0.70 0.79 0.59 0.69 Acknowledgements Thanks are due to Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi for funding the project Phosphate solubilizing microorganisms as microbial inoculants for saline area cropping system, because of which the above work has been possible. 920

Journal of Agricultural Technology 2014, Vol. 10(4): 915-922 References Abul, H.J. Ullah and Charles H.D.Jr. (1993). Identification of active site residues in Aspergillus ficuum extracellular ph 2.5 optimum acid phosphate. Biochem. and Biophy. Res. Communications, 192:754-759. Adams, A.P. (1980). In: The role of phosphorus in agriculture. (Eds) Sample E. C. and Hagimuto, I., Am. Suc. Agran. Madison, USA. pp. 655-680. Au, S., Roy, K.L. and Tigerstrom, R.G. (1991). Nucleotide sequence and characterization of the gene for secreted alkaline phosphatase from Lysobacter enzymogenes. J. Bacteriol., 173:4551-457. Dalal, R.C. (1978). Organic phosphorus. Adv. Agron., 29:83-117. Dave, A. (1999). Studies on phosphate solubilizing Pseudomonas. A Ph.D thesis, Bhavnagar. University. Furukawa, K., Hasunuma, K. and Shinohara, Y. (1987). Characterization of p i repressible enzymes secreted in cuture media by Neurospara crassa wild type cells and null type mutants. J. Bacteriol., 169:4790-4795. Goldman, S., Hecht, K., Eisenberg, H. and Mevarech, M. (1990). Extracellular Ca +2 - dependent inducible alkaline phosphatase from the extremely halophytic archaebacterium Haloarcula marismortui. J. Bacteriol., 172:7065-7070. Jackson, M.L. (1973). Soil Chemical Analysis. Prentice Hall of India Pvt. Ltd., New Delhi, pp. 134-182. McLoughlin, T., Quinn, J. and Bettermann, A. (1992). Pseudomonas cepacia suppression of sunflower wilt fungus and role of antifungal compounds in controlling disease. Appl. Environ. Microbiol., 58:1760-1763. Pederson, N.E.J. (1953). On phytin phosphorus in the soil. Plant and Soil, 4:252-266. Samaha, A.S. (1998). Studies on phosphate solubilizing yeast and filamentous fungi. Ph.D. Thesis, Bhavnagar University, Gujarat. pp. 72. Somani, L.L. and Saxena, S.N. (1971). Studies on the mineralization of organic phosphorus under the influence of crop growth in some soils of Rajasthan. J. Ind. Soc. Soil Sci., 19(3):261-267. Somani, L.L. and Saxena, S.N. (1975). Effect of some organic matter on nutrient availability, humus build-up, soil physical properties and wheat yield under field conditions. Annals of Arid Zone 14(2):149-158. Somani, L.L. and Saxena, S.N. (1976). Tropical Agriculture, 132:9-12. Somani, L.L., Bhandari, S.C., Vyas, K.K. and Saxena, S.N. (1990). Biofertilizers. Scientific Publishers, Jodhpur, India. pp. V. (Received 15 April 2014; accepted 30 June 2014) 921

Table 1. Mineralization of organic phosphates by B. sphaericus Organic sources phosphorus Sodium β glecerophosphate Days of Incubation P 2 O 5 * ph P 2 O 5 ph P 2 O 5 ph P 2 O 5 ph P 2 O 5 ph P 2 O 5 ph P 2 O 5 ph 22.44 12.98 12.21 46.23 19.81 70.88 3.8 5.5 5.2 6.4 3.9 24.52 17.95 16.28 59.53 19.01 101.59 5.3 5.8 6.4 *P solubilized as net mg % P 2 O 5 after deducting respective control. Table 2. Mineralization of organic phosphates by B. cepacia Organic phosphorus sources Sodium β glecerophosphate 27.89 11.70 15.90 62.67 20.93 69.21 7.8 5.8 5.9 6.5 25.80 61.39 37.54 78.54 19.01 53.04 7.8 5.8 5.9 6.5 28.53 68.60 46.03 118.96 19.65 25.29 3.0 8.2 6.0 6.0 6.5 29.97 73.25 71.20 34.30 20.13 61.68 2.9 8.6 6.0 6.0 6.5 35.90 74.53 110.38 24.20 26.70 53.10 Days of Incubation P 2 O 5 * ph P 2 O 5 ph P 2 O 5 ph P 2 O 5 ph P 2 O 5 ph P 2 O 5 p H P 2 O 5 ph 24.84 15.86 69.12 33.18 17.24 41.16 3.8 4.6 4.6 4.6 3.9 27.41 16.19 88.35 100.25 15.00 85.56 4.9 4.6 5.0 * P solubilized as net mg % P 2 O 5 after deducting respective control. 16.39 16.63 31.37 111.11 20.93 36.83 4.9 4.7 5.7 31.25 15.38 89.15 79.73 21.89 56.39 3.7 6.2 5.5 6.2 25.16 10.74 59.82 65.94 15.00 45.81 5.7 6.4 5.5 6.1 31.41 16.03 106.98 60.49 23.01 85.81 5.7 6.6 6.2 2 50.81 99.03 39.97 19.81 42.28 2.9 7.8 6.2 6.3 6.8 3.2 5.8 6.4 5.9 6.1 922

Journal of Agricultural Technology 2014, Vol. 10(4): 365-379